Two experiments were conducted on commercial sugar cane fields cropped with the variety SP70-1143, with the objective of evaluating a single row microplot design to determine plant recovery of 15N fertilizer nitrogen. One of them used 15N-aqua ammonia and 15N-urea applied to two linear meter microplots of a ratoon crop (four replicates). The second used one linear meter microplots (three replicates) which received 15N-aqua ammonia only. The fertilizers were applied on 15cm deep furrows, located 25cm from both sides of the cane row. One linear meter of ratoon cane, inside and outside of the microplot, and on the same and adjacent rows were harvested twelve months after fertilization. The results indicate the feasibility of using single row segments of ratoon cane with 15N-fertilizer. The main advantage of this microplot design, when compared to the classical 3 contiguous row segments, is that only one third of the labeled fertilizer is needed. In a single row, in order to separate the nitrogen taken up by plants from the fertilizer applied to the row (Nrdffr), from that applied to adjacent rows (Nrdffr+1, and Nrdffr-1), the following should be considered: (a) a border segment of 0.5 to 1.0m inside the plot, so that Ndff results from plants harvested in the center of the microplot represent the actual value of fertilizer nitrogen taken up from that applied to the same row, and (b) harvest of plants from adjacent rows at equivalent positions to those sampled inside the microplot, to quantify the 15N-fertilizer uptake by outside plants (Nr+1dffr and Nr-1dffr), which is assumed to be the same as non labeled fertilizer applied to adjacent rows (Nrdffr+l and Nrdffr-1) taken up by inside plants. The Ndfftotalvalues should be calculated by the equation: Ndfftotal = Nrdffr + Nr+1dffr + Nr-1dffr.

The use of 15N tracer opens the possibility to follow and quantify this plant nutrient in different compartments of a system under study. The main advantage is that it allows the distinction, in the plant nitrogen, between soil and fertilizer N, for instance. However, due to the high cost of the 15N labeled compounds, the size of field plots is one of the major constraints for the use of the technique.

There are examples of studies with plots varying from one single citrus plant (FEIGENBAUM et al., 1987), up to extensive areas (WENDROTH, et al, 1992) where soil spatial variability was the main focus. In most studies involving annual crops, 15N plots are small areas (microplots) with a minimum of 3 row segments, 2 to 3 meters long, placed inside larger plots fertilized at the same rate with non labeled fertilizer, used to obtain yield results. Several studies used physical barriers buried in the soil around small plots with 15N-fertilizer (SAMPAIO et al., 1984; SALCEDO & SAMPAIO, 1984b; TAKAHASHI, 1967b; WOOD, 1974). Nitrogen movement is in this way restricted to the vertical direction and lateral movement in or out of the experimental area is prevented. Although avoiding lateral N transport and reducing plot sizes and consequently 15N cost, it limits lateral root growth and causes an artificial porosity increasing water and solute movement at the physical interface (FOLLETT et al., 1991; SANCHEZ et al., 1987). If physical barriers are not used, careful attention should be given to plot size with reference to the border area of the effective harvesting area, taking into account horizontal N movement in the soil, root growth to areas outside the plot and the cost of labeled fertilizer.

The size of I5N-fertilizer microplots was evaluated for corn crop by FOLLETT et al. (1991) and for wheat by JOKELA & RANDALL (1987), OLSON (1980) and SANCHEZ et al. (1987). The authors carried out studies without physical barriers, with plots of 2 to 4m length and 1.5 to 4 m width (with 3 to 6 row segments inside).

SANCHEZ et al. (1987) presented a model of relative 15N enrichment distribution, for plants inside and outside labeled plots. If there is no preferential horizontal movement of N in the soil, plants positioned exactly on the edge of the plots (limit between plot with labeled fertilizer and outside plot with unlabeled fertilizer) should uptake half of its N from the plot having labeled fertilizer and half from outside, with non labeled fertilizer. Accordingly, they should have half of the value of nitrogen derived from the 15N-fertilizer shown by a plant located in the center of a plot of infinite size. The authors assumed that the 15N enrichment distribution for plants across a border of labeled and non labeled areas follows a sigmoidal curve, as given by equation (1) and ilustrated in Figure 1:

where Y is the the relative fraction of Ndff. Ndff(x) is the nitrogen derived from fertilizer in plants harvested at a distance (x) from the border of the labeled plot; Ndff(c) is the value for plants at the center of a plot of infinite size (plants from inside the plot with no border effect of non labeled fertilizer applied outside the plot); p is a parameter, constant for a given system, and (x) is the distance from the border (positive or negative, depending on plant position, outside or inside the labeled plot). The model was confirmed by SANCHEZ et al. (1987) and FOLLETT et al. (1991) on field trials with wheat and corn, respectively.

TAKAHASHI (1967a) used as experimental 15N fertilized plots, single sugar cane row segments, 3m long. 15N fertilizer uptake was estimated from plants of the plot and outside plot, using plot row continuation and adjacent plot rows (row up and row down). On the other hand, JOHNSON & KURTZ (1974) applied 15N labeled fertilizer on a 6m long band, midway between corn rows spaced 76cm apart, harvesting plants in adjacent rows to the labeled strip. Corn plants did not take up significant amounts of labeled N fertilizer from band more than one row away. Since the cost of isotope is one of the major expense in field experiments, the procedure of JOHNSON & KURTZ (1974) had the objective of saving 15N.

The minimum border size requirement for sugar cane fertility trials on Florida (USA) in organic soils was studied by CO ALE & SANCHEZ (1990). They applied 15N labeled fertilizer (NH4NO3) buried 3 cm beneath the surface in a 4 m long band (5cm wide), midway between two rows of sugar cane planted with 1.5m row spacing. The plant-cane and 1st-ratoon crops, at 2.25m from the tracer source had only 6% and 1% of the 15N label at the 0.75m sampling position, respectively. The authors suggested that a single border row would be sufficient for fertility trials if the levels of inter plot interference were considered tolerable.

IGUE et al. (1991) estimated size and shape of field plots for sugar cane, not including border effects in their results. Nevertheless, they pointed to the need of a better definition of plot border width.

Based on the above mentioned facts and keeping in mind the high cost of 15N-fertilizer, this research was carried out with the objective of defining an adequate use of 15N isotope methodology for the measurement of plant nitrogen fertilizer recovery by sugar cane, under brazilian field conditions. A microplot model consisting of a single cane row segment was evaluated to define both adequate sampling positions inside the plot and the calculations involved in the estimation of the total nitrogen in the plant derived from fertilizer (Ndfftotal), considering samples harvested inside and outside the plot.

MATERIAL AND METHODS

The experiments were conducted in two commercial sugar cane fields, planted with variety SP70-1143, on a dark red latosol, with less than 2% slope. The Usina Barra Grande (UBG) site (Lençóis Paulista, SP), consisted of a first ratoon crop, and the Usina São José (USJ) site (Macatuba, SP), consisted of a second ratoon crop.

The experiment at UBG contained two treatments o f nitrogen fertilizers, urea (45 % N) and aqua ammonia (18%N), at a rate of 90kg.ha-1 of N. Each treatment consisted of segments of 19 neighbouring rows of sugar cane, each 10m long and spaced 1.4m. At the center of each fourth row segment, one length of 2 m received 15N enriched fertilizer (3 atom %15N excess), resulting four plots (replicates) per treatment, located at the center of rows 4, 8, 12 and 16. The rest of the area for each treatment received equivalent amounts of non labeled fertilizer. Both treatments received 100m3. ha-1 of mixed "mosto" type vinasse, just before fertilizer application.

At USJ only aqua ammonia (18%N) was used at a rate of l00kg.ha-1 of N. The experimental area consisted of segments of 7 neighbouring rows of sugar cane, each 10m long and 1.4m apart. The centered l m of each second row received 15N-fertilizer (3 atom %15N excess), resulting in 3 plots (replicates) located at the center of rows 2, 4 and 6. The rest of the area also received non labeled aqua ammonia. Before nitrogen fertilization, 120kg.ha-1 of K2O as KC1 were applied.

Soil tillage between rows, as recommended by RODRIGUES et al. (1984), was carried out in both experiments just before fertilizer applications. The fertilizers (labeled and unlabeled fertilizers-N for UBG and USJ, and KC1 at USJ experiment) were manually distributed and buried to the 15cm soil depth in furrows spaced 25cm from both sides of all row segments.

Both experiments started in 1984. Plant shoot samples from 1m of sugar cane row were harvested after twelve months, without burning, including the trash (dry leaves). At UBG five samples per replicate, at positions A, B, C, D and E as shown in Figure 2, were taken. The average distances from the center of the plot (O) were 0.25; 0.75 and 1.25m respectively for A to C, and 1.42m for samples D and E. Samples A and B were inside the labeled plot, and samples C, D and E were outside. The experiment at USJ had a similar sampling scheme (Figure 3), resulting for each replicate, in one sample inside plot and four outside.

After measuring fresh matter yield (including trash), the plants were chopped and sub-sampled. After oven drying to constant mass at 65°C and grinding to 50 mesh size (< 0.4mm) in a Wiley mill, nitrogen content by Kjeldahl digestion-distillation (%N) and 15N abundance (atom %15N excess) by mass spectrometry (TRIVELIN et al., 1973), were performed. Plant moisture content was also determined and was used to calculate dry matter yield. The conversion factor of 7,143 (total length of rows in lha of 1.4m spaced sugar cane) was used to express the data on hectare basis. Nitrogen in the plant derived from fertilizer (Ndff), on % and kg.ha-1 was calculated from 15N abundance data (atom %15N excess) and total nitrogen in cane shoot (kg.ha-1), using the expressions:

where a e b are the 15N abundance (atom % 15N excess) of plant and fertilizer, respectively; TN is the total nitrogen accumulated in cane shoot (kg.ha-1).

As an approximation to the SANCHEZ et al. (1987) model (equation 1), theoretical values of Ndff(c) for each plot were calculated according to expression 4:

Ndff(c) is the nitrogen in the plant derived from fertilizer-N for canes from inside plot with no border effect of non labeled fertilizer applied to the row outside the plot; (e) and (-e) designate neighbour samples at symmetric positions outside and inside of the edge of the microplot, respectively.

Data were analysed using the Tukey test at 5% probability level and Student paired t test, to compare means. A linear approximation to the Sanchez'model was used. Linear regression analysis of Ndff for samples A, B and C (as dependent variable) against distances was used in both experiments to estimate the distances (xNddfc) from the center (O) of the plot where Ndff(c) values should be obtained. Thus, the theoretical border widths (Wb) could be calculated from the difference of half plot size and the distance from center, that is: Wb=1 - xNdffc, and Wb=0.5 - xNdffc, for UBG and USJ experiments, respectively.

RESULTS AND DISCUSSION

TABLES 1 and 2 show yield data for UBG and USJ, respectively. Mean values for canes at different distances from plot center (within and outside plot) did not show significant statistical differences.

The effect of the distance from plot center (canes inside and outside plot) on Ndff (% and kg.ha-1) is clear in TABLE 3. In general, Ndff decreased with distance from the center of the labeled plot, as expected. Some fluctuations are however observed, which are the result of plot length, distance from labeled border, root distribution, soil variability. Ndff for adjacent rows (row up and row down) at UBG did not differ statistically, which indicates no preferential lateral N movement in the soil. The results also show that plants from one row absorb fertilizer from adjacent rows. Therefore, when this experimental design is used, assuming no preferential lateral nitrogen movement by mass flow, the Ndff for plants of adjacent rows of the plots should be added to the Ndff for plot plants of the labelled row, when calculating total fertilizer use by the crop (Ndfftotal). For experimental plots with three or more rows of labeled fertilizer (e.g.: JOKELA & RANDALL, 1987 and FOLLETT et al., 1991), the Ndff from plants of the central row represents the total fertilizer nitrogen absorbed (from the row itself, plus adjacent rows), and it is not possible to estimate the nitrogen uptake by plants from the fertilizer applied in adjacent rows.

At UBG, the average fresh and dry matter yields, and the total nitrogen for both urea and aqua ammonia treatments (TABLE 1) were not statistically different. The same was true for Ndff for both sources of fertilizer N (TABLE 3). Those are strong evidences that sugar cane productivity and plant recovery of fertilizer N were not influenced by the nitrogen fertilizer source, as was also observed by PENNA & FIGUEIREDO (1984), and TRIVELIN et al. (1986).

Calculated values of Ndff(c)(% and kg. ha-1) are shown in TABLE 4. The values for Ndff(c) and Ndff(0.25) at UBG did not differ significantly from each other using the t test, for both urea and aqua ammonia treatments (TABLE 5). This indicates that Ndff results from the central meter of 2m long plots represent the total N uptaken by plants from the fertilizer applied to the same row. On the other hand, plants farther from the center of the labeled/plot showed significantly different Ndff values. TABLE 5 also shows that Ndff(0.25) represents at least 90% of the calculated Ndff(c) values, for aqua ammonia and urea treatments.

For the USJ experiment, Ndff(0.25) represented only 59% of the Ndff(c) and those values differed significantly by the t test (TABLE 5). In this case, all plants from the labeled plot (1m length) were sampled. Nevertheless, this plot design may be used when other samples at symmetric positions outside the microplot and close to its border are also collected.

They can be used to calculate the theoretical Ndff(c) values for fertilizer N applied to plot row, according to equation 4. This kind of microplot design can also be used in the procedures as performed by TAKAHASHI (1967a), who harvested all plot and neighbour plants, from the same and adjacent rows, to determine Ndfftotal.

The linear regressions shown in TABLE 4, all with significantly high correlation coefficients (r), were used to estimate border widths (Wb), which define the useful harvesting area, inside the microplot, to calculate Ndff(c) values. The results (TABLE 4) indicate ideal border widths on the order of 1m. On the other hand, for UBG experi-riment, 0.5m border width would be adequate for plants collected in the central 1m (TABLE 5).

The model of microplot consisting of single sugar cane row segments with 15N-fertilizer has advantages and may be used when N is banded, and only plant uptake (Ndff) is to be measured.Further studies are necessary before applying the concept to broadcast N. It should be pointed out, however, that this microplot design is not suitable to measure residual fertilizer effect or field N balance studies.

In this experiment, N uptake in sugar cane crop is given by the direct technique of determination of 15N-labeled fertilizer in the plant shoot. N uptake values by the indirect method where differences are calculated between fertilized and non fertilized treatments, are not here reported. It was found, as have others, that the direct method is both more sensitive and precise than the indirect technique in field experiments with sugar cane. Nevertheless, the 15N technique has various possible inaccuracies due to soil processes such as mineralization-immobilizationturn-over (JANSSON & PERSSON, 1982), or due the substitution of 15N by 14N, when the labeled N acts as a substitute for unlabeled soil N that otherwise would have been substracted from the pool during processes such as immobilization and denitrification (JENKINSON et al., 1985).

A complementary study would be desirable in future using a large labeled area to compare the results with the recommended 15N-fertilizer single row segments. It would be also desirable to include plots and harvests of non fertilized areas, so that the difference method could be compared.

CONCLUSIONS

The results suggest that it is possible to perform field studies using 15N-fertilizer single row segments of ratoon cane, to determine fertilizer nitrogen recovery by the crop. As an advantage, this microplot design uses one third of the labeled isotope used in conventional designs. Total plant nitrogen derived from fertilizer (Ndfftotal) can be estimated from 15N abundance in plants harvested from the same and adjacent rows, inside and outside microplots. To evaluate separately the nitrogen taken up by plants from the fertilizer applied to the row itself (Nrdffr), from that applied to adjacent rows (Nrdffr+1 and Nrdffr-1), the following has to considered: (a) a border segment of 0.5 to 1.0m length, in order to guarantee that Ndff results from plants harvested in the center of the microplot represent the maximum value of fertilizer nitrogen taken up by plants, originated from the fertilizer applied to the same row (Nrdffr); (b) harvest of plants from adjacent rows in positions equivalent to samples from inside the microplot, with the objective of quantifying the 15N-fertilizer (Nr+1dffr and Nr-1dffr) taken up by plants from outside plot, which represents the same amount of non labeled fertilizer applied to adjacent rows (Nrdffr+1 and Nrdffr-1) taken up by inside plot plants. In a field experiment the Ndfftotal value of each replicate should be calculated by the equation (5):

ACKNOWLEDGMENTS

We thank J.T. Coleti and J.C.S. Rodrigues, agronomists from Usina São José and COPERSUCAR, respectively, for their assistance in the field work.